In recent years, there has been a growing awareness of separation costs as part of the total cost in chemical processes. Sherwood (Sherwood, Pigford, and Wilke, "Mass Transfer", McGraw Hill, New York, 1975), for example, has shown a linear relationship on log-log coordinates between the value of a pure substance and the reciprocal of its concentration in the crude mixture from which it was obtained. This empirical plot suggested separation costs are often the dominant portion of the total costs of the desired product. Subsequent studies emphasizing the extraordinarily low concentrations of naturally occurring biological substances and the great difficulty to purify them have shown the correlation could be extended by several orders of magnitude using data for several differing bioproducts including materials such as interferon.
The biochemical separation processes currently used for enzyme and protein purification present further difficulties. At present, they are almost without exception highly labor-intensive, slow and relatively non-selective. A typical separation would involve gel filtration, ion exchange, or selective adsorption in chromatography columns. The fragile beads used in such columns impose pressure drop limits of 1 psi or less with correspondingly low flow rates. Another common but awkward step involves fractional precipitation of proteins followed by centrifugation and decantation. Separations based on electrical charge such as electrophoresis and isoelectric focusing offer relatively convenient ways of obtaining purified compounds at the laboratory level, but pose problems of scale up. These problems arise because the heat produced by the passage of electric current increases as the square of a dimension (i.e. as the cross section) while the surface area for heat removal goes up only linearly. Convection, band spreading, etc. also increase at high rates with increasing scale. Normally, one must stop the process and stain for proteins to see how far the separation has progressed. Attempts have been previously made to save some of the manual labor usually associated with such operations by arranging bio-separation units into a continuous processing train. Some of the units are inherently batchoriented, however, and the elaborate tour-de-force shown by these attempts serves only to enforce the notion that new purification techniques are needed.
Among the many techniques used today in biochemical separations, perhaps the most efficient and selective is one called affinity chromatography (AC). Unlike the other separation techniques mentioned, which have typical purification factors P.sub.f (=product purity/feed purity) of 2 to 10, affinity separations in favorable cases achieve P.sub.f values of 10,000 in a single step.
Unlike all of those previously mentioned and a number of others which were not mentioned, AC does not rely on general molecular properties such as size, electrical charge or density to carry out a separation. Instead, it involves a very specific interaction between two biomolecules, one of which is chemically attached to a solid support phase and the other of which is dissolved in solution (usually aqueous). Such interactions are almost a universal feature of biomolecules. Specific examples would include binding between antibodies and antigens, hormones and receptors, enzymes and either substrates, coenzymes, inhibitors or activators, DNA and its complement (a repressor or catabolite gene activator protein for double-stranded DNA or the complement of a single strand of DNA) and messenger RNA and ribosomes.
The beauty of such biochemical pairing is that since it involves a number of simultaneous interactions between amino acid or nucleotide residues, it can be highly specific. Biomolecules typically perform their functions in the presence of thousands of different types of molecules, indicating that this specificity is both a necessary and a natural part of their character. Affinity chromatography is a broad term which involves everything from a weak interaction which simply retards one molecule's passage through a column to a strong, almost nonreversible binding to the column packing. The latter would more properly be termed a bio-specific adsorption-desorption cycle. Drastic changes in pH, ionic strength, or temperature, or the addition of a competing soluble molecule are needed in such a case to release the molecule from its complement on the solid phase. This strong binding system could be operated in a batch vessel in an adsorption-desorption mode, but in most cases a column is used whether it is needed or not. Since other molecules are not usually affected by passage through the affinity column, in theory, several columns in series could be used to recover several molecules of interest from a given fermentation broth.
Despite these enormous advantages over other bioseparation schemes, affinity chromatography still has several serious disadvantages: (1) Even when operated as a column, it is a discontinuous chromatographic or adsorption-desorption process characterized by the introduction of a "pulse" of material and the recovery of a "pulse" of product. The disadvantage of this type of operation is that the size of the sample is severely limited. Most of the time the column is in operation no product is being collected, leading to an inefficient system. (2) One cannot, in such a column, use the viscous, debris-laden suspension of broken cells from a fermentation that one might hope to. A column would almost immediately plug if subjected to such a mixture. The removal of debris and DNA (whose extremely high molecular weight has a large effect on viscosity) is still a serious problem in industrial-scale processes. (3) Since peak emergence from the column is related to time, control and automation of the process is more difficult than it is for a steady-state operation.
Recognizing these shortfalls, attempts were made to overcome these problems by devising various types of continuous chromatographic techniques. The aim was to eliminate the inefficiency of a batch operation by allowing the sample to be injected continuously and the products to be continuously withdrawn. These techniques utilized a moving chromatographic bed wherein the movement (or in some cases a simulated movement) in each case is either perpendicular to the solvent flow, allowing a number of different compounds to be purified simultaneously, or countercurrent to the flow, in which case usually only two pure components are obtained. The advantage of either variation is the relatively high throughput which can be obtained compared to repeated batch operations. The disadvantage of some of these techniques, such as the simulated moving bed, is that they require elaborate and expensive mechanical moving seals or automatic valves to operate. In addition to the added expense, the risk of contamination is high when the system is one involving biomaterials, and when it is operated over long periods of time. Also, the problem of clogging by debris is not eliminated by any of these continuous systems.
A recent development which might be used to advantage to eliminate or substantially reduce the problem of clogging while retaining the other advantages of continuous chromatography is the magnetically stabilized fluidized bed. The ordinary fluidized bed has been used in industrial processing for many years, mostly with catalytic particles which tend to foul or become poisoned or where thermal effects are important. Above a certain critical fluid velocity, small particles of a solid become suspended in a high velocity stream and the solids suspension acts much like a fluid, permitting it to flow out of the reactor for regeneration or replacement. If the fluid velocity is increased above the critical fluidization value, undesirable effects such as bubbling and slugging occur. These cause bypassing of reactants through the bed and can result in particle entrainment in the gas. Although these problems are less severe in beds fluidized with liquids rather than with gases, the fluidized particles still undergo a strong back-mixing process so that the bed behaves much like a continuous flow stirred-tank reactor. Although this turbulence may be desirable for certain processes such as heat exchange, it would be highly detrimental to any type of chromatographic separation.
As early as 1961, Hershler experimented with magnetic fields applied to liquid metals and magnetically susceptible solids which had been fluidized. He reported in the patent literature (U.S. Pat. Nos. 3,219,318 and 3,439,899) that a magnetic field created with an alternating current could be used to stir such liquid metals, fluidize beds even in the absence of a supporting gas or liquid stream, and (with several isolated fields in a column) decrease the bubbling and prevent material from being ejected from the top of a fluidized bed. The mechanisms of these actions apparently were not investigated to any great extent, and it is clear from the drawings in these patents that the magnetic fields were far from uniform.
Other work on magnetic fields in conjunction with fluidized beds was carried out by Tuthill (U.S. Pat. No. 3,440,731), however, it was not until the late 1970's when Rosensweig began publishing in this area that careful and systematic study of magnetically stabilized fluidized beds began ("Magnetic Stabilization of the State of Uniform Fluidization, Ind. Eng. Chem. Fund., 18:260; "Fluidization: Hydrodynamic Stabilization With A Magnetic Field", Science, 204:57; and with Lucchesi, Hatch, and Mayer, "Magnetically Stabilized Fluidized Beds", A.I.Ch.E. Symp. Series 77, #205, 8). Among the important findings of Rosensweig and his co-workers are these: First, fluidization of magnetically susceptible solids can be stabilized in a uniform gradientless magnetic field in which the individual particles experience no net force. An axially-oriented field is preferred, although the orientation of the field is not crucial. Second, stabilization is observed over a wide range of field strengths and fluidization velocities, and the applicable ranges of the important variables have now been mapped out by Rosensweig. For most fluid velocities, when the bed is stabilized, a decrease in magnetic field strength will result in normal fluidization while an increase will result in agglomeration of the solid particles. The effect of the magnetic field can be viewed roughly as creating a magnetic dipole in each particle which causes it to become "sticky" in a direction parallel to the field lines. This produces what amounts to chains of beads parallel to the axis of the bed.
As is the case in a ordinary fluidized bed, the particles in a magnetically stabilized fluidized bed behave as a fluid over a wide range of conditions. Their apparent density is greater than the fluid phase but less than the actual solid density. Unlike the ordinary fluidized bed, however, the dispersion and back-mixing of particulates is effectively zero. The magnetically stabilized fluidized bed is therefore an extremely interesting new phenomenon in its own right and is worthy of considerable further basic study. In addition, however, the properties of a magnetically stabilized fluidized bed are ideal for use in a continuous chromatography system. In this application, the fluid-like behavior of the solids would allow countercurrent solids/solvent contacting. Clogging by debris should be controllable, because the bed contents, along with debris that they filter out, can be continually removed and replaced. All of these factors suggest chromatography in a magnetically stabilized fluidized bed would be a highly efficient separation scheme, and particularly in bioseparations because of the great need for improved processing of biomaterials; a fairly complex scheme such as this is most easily justified for products which have a high dollar value per pound. Prior to the present invention, however, the use of such technology has not been applied to these separations.
An examination of the support media presently available for use in a magnetically stabilized fluidized bed separation of biomaterials, however, was not successful. Prior to the present invention, there were no magnetic particles available which met the requirements for bioseparations, specifically these requirements of high density, accurate sphericity and uniform size, low porosity, and a high concentration of chemical groups which could be used to bind the affinity ligand through standard immobilization reactions. Metals such as nickel were lacking the last characteristic, and commercial composite materials were too low in density and too porous. The various requirements just listed were arrived at through a series of theoretical predictions and practical tests. In brief, they can be summarized by stating that the high density and moderately large size were necessitated by the use of a relatively dense and viscous fluidization phase, for example, water. The large size in turn dictated low porosity to prevent undesirable chromatographic band spreading from intra-particle diffusion delays. Finally, a nonporous particle demands a high concentration of surface binding sites so that its adsorbing capacity is acceptably high.
Calcium alginate gels have been previously used as a biomaterial support for many different immobilized enzyme and cell preparation systems. The support is biochemically inert, easy to handle, and can be packed, like any other gel, into affinity chromatography columns. Immobilization (the techniques for which have been reported extensively) is usually accomplished by entrapment; the desired enzyme or cell population is mixed with the alginate solution and, upon polymerization, is "trapped" in the gel matrix. The gel itself offers little resistance to substrate diffusion.
For a number of reactions and separation systems, however, diffusion of reactants into the interior of the support is either undesirable or impossible. Enzymes which react with large substrate molecules are wasted if they are immobilized in regions of the gel where the substrate cannot penetrate. Affinity cell separation systems which contain ligand dispersed in the support are likewise inefficient, since the cells only contact the surface of the gel. Systems such as these would be more efficient with the reactive species coupled only to the bead's surface.
Some separation techniques now being used also require magnetic supports to operate efficiently. High gradient magnetic filtration, for example, is one such technique which allows both filtering of lysed cell parts and purification of the enzyme being sought. In this technique the support with an affinity matrix attached is added to the disrupted cell mixture. The solution and support are then passed through a high gradient magnetic filter where the magnetic support is retained but the insoluble proteins and debris continue through. The field is then removed and the purified enzyme is obtained after desorption from the support. The supports used in the past for such separations have been metals or various gels with magnetic particles either adsorbed on their surface or dispersed throughout the gel matrix.
The support described in the present invention offers a new application of alginate in the biotechnology field. Although similar in some ways to others currently available, the beads have unique and highly desirable features. Alginate, the polymeric material from which the beads are made, is a block copolymer extracted from kelp consisting of .beta.-D-mannuronate (M) and .alpha.-L-guluronate (G) residues. Exposure to calcium ions in solution crosslinks the acid residues of the alginate molecules into a gel, producing a fairly stable support. When particles of magnetite (Fe.sub.3 O.sub.4), a magnetic oxide of iron, are mixed with the alginate solution before gelation (a generalization of the process disclosed herein), the beads change from a cloudy white support to an opaque black magnetic support. When the beads are dried, the support shrinks irreversibly from the hydrogel state to a rigid solid while remaining quite spherical and highly magnetically susceptable. The density of the dried support is on the order of glass, but the reactivity is considerably greater. The porosity of the support is limited, but the exposed surface is microscopically very rough, providing many sites for protein or cell attachment.
It is, therefore, an object of the present invention to disclose a novel magnetic chromatographic separation support material.
It is further object of the present invention to disclose the use of a novel magnetic support material in affinity chromatography of bioproducts.
It is still a further of object of the present invention to disclose affinity chromatography of bioproducts carried out in a magnetically stabilized fluidized bed.
The following description of the drawing and examples are presented in order to allow for a more thorough understanding of the subject matter and experimental procedure of the present invention. The drawing and examples are meant to illustrate the embodiments of the present invention, and are not to be construed as limiting the scope of the invention.